# US 8,772,460 B2 · Thermostable FGF-2 mutant having enhanced stability

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- Number: US 8,772,460 B2
- Document ID: US8772460B2
- Status: Granted
- Kind: B2
- Application number: 13/717,275
- Filed: 2012-12-17
- Published: 2013-09-12
- Granted: 2014-07-08
- Earliest claimed: 2011-12-16
- Assignees: Wisconsin Alumni Research Foundation
- Applicants: Wisconsin Alumni Research Foundation
- Inventors: Guokai Chen; James A. Thomson; Zhonggang Hou
- Web page: https://patentgrep.com/patent/US8772460B2

## CPC

- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- C07K 14/503: {basic FGF [bFGF]}
- C12N 5/0607: {Non-embryonic pluripotent stem cells, e.g. MASC (induced pluripotent stem cells C12N5/0696)}
- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/91: Heparin
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Thermostable FGF-2 proteins having enhanced ability to support human pluripotent stem cell cultures are provided. Also provided are methods and compositions utilizing thermostable FGF-2 proteins.

## Claims

### Claim 1

An isolated fibroblast growth factor-two, FGF-2, polypeptide, as depicted in SEQ ID NO: 2, that differs from wild type FGF-2, as depicted in SEQ ID NO: 1, at amino acid position 128, wherein the isolated FGF-2 contains an asparagine submitted for lysine at position 128 (a K128N substitution).

### Claim 2 (depends on 1)

An isolated nucleic acid encoding the isolated FGF-2 of claim 1.

### Claim 3 (depends on 1)

An isolated genetically modified cell producing the isolated fibroblast growth factor of claim 1.

### Claim 4

A method for culturing human pluripotent stem cells, the method comprising the step of culturing a human pluripotent stem cell in a medium comprising a K128-substituted thermostable fibroblast growth factor-two, FGF-2, as depicted in SEQ ID NO: 2, that differs from wild type FGF-2, as depicted in SEQ ID NO: 1, at position 128 of the wild type FGF-2, wherein the thermostable FGF-2 contains an asparagine submitted for lysine at position 128 (a K128N substitution).

### Claim 5 (depends on 4)

The method of claim 4, wherein the human pluripotent cells are selected from the group consisting of human embryonic stem cells and human induced pluripotent stem cells.

### Claim 6 (depends on 4)

The method of claim 4, wherein the medium further comprises heparin.

### Claim 7 (depends on 4)

The method of claim 4, wherein the medium comprises the thermostable FGF-2 at a concentration of less than 40 ng/ml.

### Claim 8 (depends on 7)

The method of claim 7, wherein the medium comprises the thermostable FGF-2 at a concentration less than 10 ng/ml.

### Claim 9 (depends on 8)

The method of claim 8, wherein the medium comprises the thermostable FGF-2 at a concentration less than 3 ng/ml.

### Claim 10 (depends on 9)

The method of claim 9, wherein the medium comprises a thermostable FGF-2 at a concentration less than 1 ng/ml.

### Claim 11

A fully defined culture medium suitable for culturing human pluripotent cells in an undifferentiated state, the medium comprising thermostable fibroblast growth factor-two, FGF-2, as depicted in SEQ ID NO:2, that differs from wild type FGF-2, as depicted in SEQ ID NO: 1, at amino acid 128 of the wild type FGF-2, wherein the thermostable FGF-2 contains an asparagine submitted for lysine at position 128 (a K128N substitution).

### Claim 12 (depends on 11)

The fully-defined medium of claim 11, wherein the fully defined medium comprises thermostable FGF-2 at less than 40 ng/ml.

### Claim 13 (depends on 12)

The fully-defined medium of claim 12, wherein the fully defined medium comprises thermostable FGF-2 at less than 10 ng/ml.

### Claim 14 (depends on 13)

The fully-defined medium of claim 13, wherein the fully medium comprises thermostable FGF-2 at less than 3 ng/ml.

### Claim 15 (depends on 14)

The fully-defined medium of claim 14, wherein the concentration of the thermostable FGF-2 in the fully defined medium is equal to or less than 1 ng/ml.

### Claim 16

A composition comprising:
a human pluripotent stem cell;
a medium suitable for culturing the human pluripotent cell in an undifferentiated state; and
a thermostable fibroblast growth factor-two, FGF-2, as depicted in SEQ ID NO:2, that differs from wild type FGF-2, as depicted in SEQ ID NO: 1, at amino acid 128 of the wild type FGF-2, wherein the thermostable FGF-2 contains an asparagine submitted for lysine at position 128 (a K128N substitution).

### Claim 17 (depends on 16)

The composition of claim 16, wherein the medium comprises thermostable FGF-2 at less than 40 ng/ml.

### Claim 18 (depends on 17)

The composition of claim 17, wherein the medium comprises thermostable FGF-2 at less than 10 ng/ml.

### Claim 19 (depends on 18)

The composition of claim 18, wherein the medium comprises thermostable FGF-2 at less than 3 ng/ml.

### Claim 20 (depends on 19)

The composition of claim 19, wherein the medium comprises thermostable FGF-2 at less than 1 ng/ml 1.

### Claim 21

A method for culturing reprogrammed human somatic cells comprising:
culturing reprogrammed human somatic cells in a medium until they express markers indicative of induced pluripotent stem cells, wherein the medium comprises water, salts, amino acids, vitamins, a carbon source, insulin, selenium and thermostable fibroblast growth factor-two, FGF-2, as depicted in SEQ ID NO:2, that differs from wild type FGF-2, as depicted in SEQ ID NO: 1, at amino acid 128 of the wild type FGF-2, wherein the thermostable FGF-2 contains an asparagine submitted for lysine at position 128 (a K128N substitution).

### Claim 22 (depends on 21)

The method of claim 21, wherein the medium comprises thermostable FGF-2 at less than 40 ng/ml.

### Claim 23 (depends on 22)

The method of claim 22, wherein the medium comprises thermostable FGF-2 at less than 10 ng/ml.

### Claim 24 (depends on 23)

The method of claim 23, wherein the medium comprises thermostable FGF-2 at less than 3 ng/ml.

### Claim 25 (depends on 22)

The method of claim 22, wherein the medium comprises thermostable FGF-2 at less than 1 ng/ml.

### Claim 26 (depends on 21)

The method of claim 21, wherein the medium is serum-free.